Role of mitochondrial dynamics and function in Drosophila embryo morphogenesis

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Role of mitochondrial dynamics and function in Drosophila embryo

morphogenesis

A thesis

Submitted in partial fulfilment of the requirements of the degree of

DOCTOR OF PHILOSOPHY by

SAYALI CHOWDHARY 20112001

INDIAN INSTITUTE OF SCIENCE EDUCATION AND RESEARCH

PUNE

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Dedicated to my beloved Aai, Baba and Aaji

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Certificate

It is certified that the work incorporated in the thesis entitled ("Role of mitochondrial dynamics and function in Drosophila embryo morphogenesis") submitted by Sayali Chowdhary was carried out by the candidate, under my supervision. The work presented here or any part of it has not been included in any other thesis submitted previously for the award of any degree or diploma from any other University or institution.

Date: 19/03/2019 Dr. Richa Rikhy

(Supervisor)

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Declaration

I declare that this written submission represents my research work in my own words and where others’ ideas or works have been included, I have adequately cited and referenced the original sources. I also declare that I have adhered to all principles of academic honesty and integrity and have not misrepresented or fabricated or falsified any

idea/data/fact/source in my submission. I understand that violation of the above will be the cause for disciplinary action by the Institute and can also evoke penal action from the sources which have thus not been properly cited or from whom proper permission has not been taken when needed.

Date: Sayali Chowdhary

Reg. No: 20112001

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Acknowledgements

It has been a great learning experience since I joined IISER, Pune for Integrated PhD

program. It would not have been possible for me without the support I received from many people during this journey.

I am extremely grateful to my PhD thesis advisor Dr. Richa Rikhy for giving me an opportunity to work with her. She has always been enthusiastic about new ideas, encouraged me to come up with “jugaads” and helped me grow professionally and personally. She has always given her sincere word of advice, whether about science or otherwise. I thank her for sharing her immense expertise in genetics and imaging

throughout. She has always encouraged me to attend relevant meetings and discuss ideas with eminent scientists.

Thanks to my RAC members Dr. Aurnab Ghose, Dr. Nagaraj Balasubramanian and Dr. Deepa Subramanyam for their criticism and experimental suggestions during the RAC meetings. I would also like to thank Dr. Girsh Ratnaparkhi for questions and suggestions during the lab meetings. His questions and wisdom about the experimental basics helped in revisiting the project in different angles.

Dr. Nagaraj Balasubramanian was my mentor for the first two years of integrated PhD. He helped me a lot in getting adjusted to the system and was patient in answering my

questions about the coursework and overall PhD structure.

I thank Rachel Cox, Thomas Lecuit, Leo Pallanck, Krishanu Ray, Siegfried Roth, Anuradha Ratnaparkhi and Girish Ratnaparkhi for Antibody and fly reagents used in the project. I also thank BDSC, VDRC and NCBS stock centres for fly stocks.

IISER, Pune provides state of the art infrastructure with incredible facilities. I would like to thank fly facility (Snehal, Yashwant and others) for maintaining stocks and providing fly media, microscopy facility (Vijay, Santosh, Rahul and Aditi) and administration (Mrinalini, Piyush, Kalpesh, Shabnam, Roopali, Mahesh, Nayana and Tushar). I am grateful to Vijay Vittal for teaching microscopy basics and help with imaging troubleshooting.

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Special thanks to University Grants Commission for my graduate fellowship. Additionally, I thank Infosys Foundation and Department of Biotechnology, India for funding conference travel expenses.

I would like to thank Bhavin, Somya, Bilwa, Dnyanesh, Debasmita and Akshada for their help in weeding out errors and typos in some of the thesis chapters.

RR lab members is a super crazy bunch of people who appropriately match to the lab acronym M.A.D. lab. All of my labmates have been extremely helpful, especially in changing fly cage plates many times for me. Lab seniors Aparna and Darshika taught experimental techniques during lab rotation. Bipasha has always offered her selfless help during any experiment. Sameer's technical queries durin g lab meetings helped in improving analysis.

Dnyanesh and Bhavin's suggestions about various experiments were useful. Swati has always given an ear to my random rants and shared her life philosophy whenever I needed to get focused. I am thankful to Darshika and Dnyanesh for their help with the manuscript.

Thanks to Devashree for cloning Mito-PA-GFP line. I would like to thank previous lab members: Tirthasree, Vishnu, Radhika, Rohan, Prachi, Gayatri and Prachiti for their

suggestions and help. It was a great experience working with Arijit, Abhijeet, Bhagyashree D.

and Somya. Bhagyashree helped with analysis of drp1^SG; opa1ⁱ data during her lab rotation.

Somya helped in setting up experiments for Toll-Dorsal pathway modifications. I thank all the lab members and Richa for maintaining a cheerful and optimistic work environment in the lab.

I am sure to have found friends here at IISER during these years of integrated PhD. I would like to thank my batchmates for being “co-sufferers”. Thanks to Libi, Bhagyshree, Rupa and Manawa for their support and words of wisdom. Ketakee, Chaitanya, Sampada, Shubhankar, Aditi, Niraja, Devika, Sanket, Shraddha and Manasi were greatly successful in distracting me from work. Thanks to annoying little kids: Gayatri and Prachiti for putting up with my craziness. I have found sisters for life in these two. Evening coffee sessions with Ketakee, Chaitanya, Sampada and Aditi would turn a 10 mins break into an hour long discussion session over any given topic ranging from science to philosophy and comics to real world issues. I have shared numerous crazy moments with Aditi, Sampada and Ketakee which I will cherish forever.

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Thanks to coffee, coffee and some more coffee from G1 canteen and CCD for keeping me awake during long sessions. I also thank G1 canteen Anna, for providing evening snacks and a humorous statement for free with the food.

I am thankful to my non-IISER friends for their constant support and boost. Spontaneous gossip and discussion sessions with Bilwa, Radhika, Loukik, Tanvee, Prashant, Akshada, Mugdha and Achintya have helped in reliving the work stress.

I am lucky to have an incredibly supportive family. Thanks to aai, baba and aaji who have encouraged and nurtured my dream to become a scientist and being strong pillars of support throughout. No words can express my gratitude for them.

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7.3.1 Drp1 knockdown affects ventral furrow formation 143 7.3.2 Distribution of Dorsal and Twist is unchanged in drp1^SG embryos 145 7.3.3 Increased Dorsal elevates apical mitochondria during gastrulation 146 7.3.4 Mitochondria accumulate at the ventral furrow in rhogefⁱand mbsⁱ 149 7.3.5 fog knockdown reduces mitochondrial apical transport in the

ventral furrow 150

7.4 Discussion 152

8 Thesis Summary and Future perspectives 155

8.1 Regulation of mitochondrial distribution during Drosophila embryogenesis 156 8.2 Role of mitochondrial shape and function during embryogenesis 157 8.3 Regulation of mitochondria by Toll-Dorsal pathway 159

8.4 Experimental limitations 163

8.5 Future perspectives 164

9 Appendices 168

A1 Comparison of Mitochondria, ER and Golgi Complex localization in the

syncytial Drosophila embryo 168

A2 Interaction of mitochondria with ERK signalling in Drosophila

embryogenesis 170

10 Bibliography 172

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vi

List of Figures

Chapter 1

1.1 Mitochondrial structure

1.2 ATP is generated by mitochondrial electron transport chain 1.3 Mitochondria undergo fusion and fission

1.4 Physiological roles of mitochondrial morphology 1.5 Mitochondrial transport of microtubules

1.6 Drosophila embryogenesis as a model system to study mitochondria Chapter 2

2.1 Knockdown of genes in the embryos using maternally expressed Gal4 Chapter 3

3.1 Schematic representing NCs during syncytial stage of Drosophila embryogenesis 3.2 Mito-GFP colocalizes with streptavidin

3.3 Mitochondrial distribution in preblastoderm embryos 3.4 Mitochondrial distribution in syncytial blastoderm embryo

3.5 Mitochondria show restricted movements in the lateral plane of syncytial cells 3.6 Mitochondria are symmetrically distributed to syncytial daughter cells

3.7 Apical mitochondrial number increases during metaphase of the syncytial cycle 3.8 : Microtubules regulate mitochondrial distribution in syncytial embryos

3.9 Mitochondrial localization in pole cells Chapter 4

4.1 CMXRos staining after ETC inhibition in Drosophila embryos 4.2 Inhibition of ETC depletes ATP in the syncytial Drosophila embryo 4.3 pAMPK localization in syncytial NCs

4.4 ETC inhibition elevates pAMPK signal in syncytial embryos 4.5 Total AMPK levels do not change on genetic depletion of ETC

4.6 Western blotting of pAMPK and total AMPK in WT, pdswⁱ and covaⁱ embryos 4.7 Mitochondrial distribution in genetic inhibition of ETC

4.8 : Inhibition of ETC decreases metaphase furrow extension in syncytial Drosophila embryos

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vii 4.9 Inhibition of glycolysis does not affect pAMPK levels and metaphase furrow length

Chapter 5

5.1 Schematic representing cellularization and gastrulation stage of Drosophila embryogenesis

5.2 Mitochondria migrate apically during cellularization 5.3 Mitochondrial distribution during gastrulation

5.4 Apical migration of mitochondria is regulated by microtubules

5.5 Acto-myosin dynamics is not essential for mitochondrial apical migration Chapter 6

6.1 Marf and Drp1 localize on the mitochondria

6.2 Drp1 mutant embryos have clustered mitochondria

6.3 Mitochondrial morphology does not change in fusion protein knockdown

6.4 Apical migration of mitochondria during cellularization is abolished in Drp1 KD embryos 6.5 Shorter cells are formed in drp1SG embryos

6.6 Contractility of actomyosin rings is reduced in drp1SG cellularizing embryos 6.7 ATP and ROS measurements in drp1SG

6.8 Mitochondrial migration and contractile ring area are rescued in drp1SG; opa1i embryos 6.9 Mitochondrial ETC does not affect mitochondrial transport

Chapter 7

7.1 drp1 mutant embryos have misaligned ventral furrow cells with lowered Myosin (Sqh) 7.2 Distribution of Dorsal and Twist in unaffected in drp1SG embryos

7.3 Mitochondrial apical migration is enhanced in dorsal over-expression 7.4 Downregulation of Toll-Dorsal pathway components

Summary and Future perspectives 8 Summary of results

Appendix 1

A1.1 Comparison of mitochondria with ER and Golgi Complex distribution Appendix 2

A2.1 Levels of dpERK signal are elevated in drp1^SG embryos

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viii

List of Tables

Chapter 2

2.1 List of Drosophila stocks used

2.2 List of antibodies and fluorescent probes 2.3 List of inhibitors and fluorescent dyes Chapter 3

3.1 Fly Strains used 3.2 List of reagents Chapter 4

4.1 List of fly stocks 4.2 List of reagents Chapter 5

5.1 List of fly stocks 5.2 List of reagents Chapter 6

6.1 List of fly stocks 6.2 List of reagents

6.3 Phenotype standardization of various drp1 knockdown strategies 6.4 Phenotype standardization of various fusion knockdown strategies Chapter 7

7.1 List of fly stocks 7.2 List of reagents

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Abbreviations

2-DG 2-deoxy-D-glucose

AMPK 5' adenosine monophosphate-activated protein kinase ATP Adenosine Tri Phosphate

Bcl-2 B-cell lymphoma 2

CoVa Cytochrome C oxidase subunit Va

Cta Concertina

Dhc64C Dynein Heavy Chain 64C subunit Dhc Dynein Heavy chain

DHE dihydroethidium

Drp1 Dynamin related protein 1

EGFR Epidermal Growth factor receptor EM Electron microscopy

ER Endoplasmic Reticulum

ERK Extracellular signal regulated kinases ESCs Embryonic Stem Cells

ETC Electron Transport Chain

FCCP Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone FRAP Fluorescence Recovery after Photobleaching

FLIP Fluorescence Loss in Photobleaching FAD Flavin adenine dinucleotide

Fog Folded Gastrulation

FOXO1 Forkhead box, sub-group O

Fzo Fuzzy Onions

GSK3 Glycogen Synthase Kinase 3 HIF-α Hypoxia induced factor α JNK c-Jun N-terminal kinase Khc Kinesin Heavy Chain LH Limonene:Heptane (1:1) Lkb1 Liver kinase B1

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x MAM Mitochondria Associated ER Membrane

Marf Mitochondrial assembly regulatory factor Mff Mitochondrial fission factor

Mfn Mitofusin

Mdv1 Mitochondrial division protein 1 MRLC Myosin Regulatory Light Chain Miro Mitochondrial RhoGTPase mtDNA Mitochondrial DNA

mtlrRNA Mitochondrially encoded large ribosomal RNA mTOR mammalian target of rapamycin

NADH Nicotinamide adenine dinucleotide NC Nuclear division cycle

NF-κB Nuclear factor-kappaB PA Photo Activation

PBS Phosphate buffered saline

PDSW NADH dehydrogenase (ubiquinone) PDSW subunit PFA para-formaldehyde

PGC Posterior Germ Cells

OMM Outer mitochondrial membrane Opa1 Optic atrophy 1

ROI Region of Interest ROS Reactive Oxygen Species SEM Standard error of mean SOD Superoxide dismutase TAG Triacyl Glycerides TCA Tricarboxylic Acid Cycle

TMRE tetramethylrhodamine, ethyl ester wntD wnt inhibitor of Dorsal

WT Wild Type

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Abstract

Mitochondria, in addition to their well-known function of ATP synthesis, are also involved in a wide variety of cellular processes, such as apoptosis, calcium homeostasis and signalling.

Mitochondria are structured by dedicated fusion and fission machinery that consists of well characterized GTPase proteins. The mitochondrial morphology is modified based on cell type and physiological requirements and is linked with a wide range of signalling pathways.

Embryogenesis involves complex cascades of signalling that occur at specific milestones of the development. Hints from the literature suggest a temporal regulation of mitochondrial function and localization in ascidian and mammalian embryos. Mislocalization and

deregulation of mitochondrial structure lead to cellular and embryonic lethality. However, a systematic analysis of mitochondrial shape and localization with respect to functional regulation and signalling during embryogenesis has not been carried out so far. We characterized mitochondria in early Drosophila embryogenesis using mitochondrially localized GFP and found that mitochondria are small and dispersed around nuclei in

syncytial, cellular blastoderm, and gastrulating embryos. Mitochondria are basally enriched during syncytial stage and are actively re-distributed to the apical side during cellularization.

Apical migration of mitochondria is specifically enhanced in the ventral furrow cells during gastrulation. This re-localization of mitochondria is microtubule dependent. This apical redistribution of mitochondria is abolished in embryos mutant for mitochondrial fission protein Drp1. Myosin II levels are reduced in drp1 mutant embryos and the cells formed during cellularization in embryos are shorter and have wider contractile rings at their basal regions. The misaligned ventral furrow cells also have lowered Myosin II accumulation at their apical regions in drp1 mutant embryos. This is likely due to reduced levels of reactive oxygen species (ROS) in these embryos. We find that apical mitochondrial transport in ventral furrow cells is regulated by the Toll-Dorsal pathway. Upregulation of Dorsal

enhances the ventral signalling and apical accumulation of mitochondria whereas fog loss of function shows reduced mitochondrial apical transport in the ventral furrow cells. Thus, we demonstrate that mitochondrial localization is regulated by the Toll-Dorsal pathway and propose that their function is essential for functioning of Toll-Dorsal pathway. We are further examining the functional role of mitochondria in regulation of this pathway.

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Synopsis

Name of the Student: Sayali Chowdhary Registration number: 20112001

Name of Thesis advisor: Dr. Richa Rikhy Date of Registration: 1st August 2011

Place: Indian Institute of Science Education and Research, Pune

Title: Role of mitochondrial dynamics and function in Drosophila embryo morphogenesis

1. Introduction

Mitochondria are primarily known for their function in ATP synthesis and thereby called

“Power house of the Cell”. In addition to this prime function, they are also involved in regulation of a number of physiological processes such as calcium signalling and ROS production. Mitochondrial shape is dynamic in the eukaryotic cells and they can exist as an intricate reticular network or small punctate form (Bereiter-Hahn and Vöth, 1994). The mitochondrial structure is often correlated with their function and metabolic output (Mishra and Chan, 2016). Larger mitochondria have a complex cristae organization which helps harbouring more electron transport chain (ETC) components (Cogliati et al., 2013) thereby enhancing the ATP output in differentiated cells such as muscles, neurons, pancreatic cells and cardiomyocytes (Kuznetsov et al., 2009). On the other hand small mitochondria are considered to be poor ATP producers in the literature and are present in stem cells and embryonic cells (Lees et al., 2017; Motta et al., 2000; Sathananthan and Trounson, 2000).

Mitochondrial shape is regulated by fission and fusion proteins with aid from the cytoskeleton. These proteins are dynamin family GTPases which can actively modify mitochondrial membrane architecture (Chan, 2006). Different mitochondrial shapes are associated with a variety of signalling pathways. Mitochondrial fragmentation by EGFR pathway is essential for cellular differentiation (Mitra et al., 2012; Tomer et al., 2018).

Regulation of mitochondrial fusion by Hippo/Yorkie pathway is implicated in cell growth and cancers (Nagaraj et al., 2012). Mitochondrial interaction with Bcl-2 family proteins is

essential for apoptosis (Dewson and Kluck, 2009). Mitochondrial size is regulated during cell cycle to provide for increase energy demands (Mitra et al., 2009). More such emerging

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xiii studies indicate involvement and regulation of mitochondria in a number of key signalling pathways of cell fate determination and patterning based on their structure and dynamics.

Embryogenesis consists of cascades of complex signalling that are spatio-temporally regulated as the embryonic stem cells specialize to attain a peculiar fate. They are maternally inherited in the embryos and therefore implicated in diseases related to mitochondrial DNA (mtDNA) mutations (Giles et al., 1980; May-Panloup et al., 2016).

Mitochondria are small and dispersed in the early blastoderm embryos (Acton et al., 2004;

Van Blerkom et al., 2000; Motta et al., 2000; Sathananthan and Trounson, 2000) and their ATP synthesis is initiated by Calcium signalling during fertilization of the eggs (Dumollard et al., 2003; Roegiers et al., 1995). Although they are abundant around the nuclei, the active mitochondria are localized cortically in the mammalian embryos (Acton et al., 2004; Van Blerkom et al., 2003). Mitochondrial distribution is asymmetric in Xenopus and sea urchin embryos (Roegiers et al., 1995). Mitochondrial asymmetric distribution and activity define the oral-arboral axis in the sea urchin embryos (Coffman et al., 2004). Another study

postulates increased mitochondrial localization and function at the prospective gastrulation site in Xenopus embryos (Yost et al., 1995). A thorough analysis of how mitochondria may regulate processes during embryogenesis is absent in the field. This led us to perform a systematic analysis of mitochondrial morphology and function in the embryonic

development.

We characterized mitochondrial morphology and distribution in the early Drosophila embryogenesis and attempted to understand the effects of mitochondrial morphology alteration at the cellular level. The key findings are as follows:

2. Results

2.1 Mitochondria are enriched basally and are compartmentalized in the syncytial cells Nuclei in the syncytial embryo are partially covered with plasma membrane on the apical regions. Despite the presence of free cytoplasm at the basal regions, organelles such as ER and Golgi and plasma membrane associated proteins localize within one nuclear-

cytoplasmic territory (Frescas et al., 2006; Mavrakis et al., 2009). We visualized

mitochondria using maternally expressed mitochondrially localized GFP (Mito-GFP) in the

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xiv syncytial embryos and found that they occurred as small discrete structures enriched at the basal regions of syncytial cells. The basal enrichment of mitochondria is determined by microtubule motor Kinesin-1. We monitored their movement using photobleaching and photoactivation. The mitochondria did not have significant lateral movement, leading to their restricted localization within a syncytial cell.

2.2 Mitochondrial metabolism is required for metaphase furrow elongation.

First, to test the mode of metabolism in the Drosophila embryos, we inhibited either glycolysis or ETC using pharmacological inhibitor drugs and measured the levels of ATP sensor activated AMPK (pAMPK) in the syncytial cells. AMPK is activated by phosphorylation by upstream kinases upon ATP depletion (Hardie et al., 2006; Sakamoto et al., 2005).

pAMPK signal in the syncytial cells did not change upon inhibition of glycolysis and was significantly elevated in the treatments with ETC drug inhibitors. Similar results were obtained when we knocked down components of ETC: PDSW subunit of NADH

dehydrogenase complex (Complex I, pdsw) and cytochrome C oxidase subunit Va (Complex IV, cova), using RNAi based approach. Thus these data established that mitochondrial ETC is the ATP source in the syncytial embryos.

We then explored the implications of ATP depletion. The short membrane structures

covering the syncytial nuclei extend basally during metaphase forming furrows with the help of rapid actin assembly and may require abundant energy supply. The treatment with ETC inhibitor drugs and knockdown of pdsw and cova led to shortening of these metaphase furrows. Therefore despite the small shape, mitochondria produce ATP, which may be locally delivered to the growing membranes for rapid actin dynamics. It also possible that the phenotype of shorter furrows is due to interaction of AMPK signalling with cytoskeletal components (Cook et al., 2014; Lee et al., 2007).

2.3 Mitochondria are transported apically during cellularization and gastrulation

During cellularization the short membranes extend towards basal regions and enclose the nuclei to form tall epithelial cells (Mazumdar and Mazumdar, 2002; Warn and Robert- Nicoud, 1990). We imaged mitochondria during cellularization using the Mito-GFP tag and observed their increased accumulation at the apical regions of cells. Using photoactivation

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xv we showed that they migrate apically from basal regions. The apical migration is assisted by microtubule motors. Knockdown of Kinesin-1 (khc) resulted in premature mitochondrial transport toward the apical regions whereas they clustered at the basal regions in Dynein (dhc) knockdown embryos. Thus Dynein motors transport mitochondria apically during cellularization.

To observe mitochondrial dynamics during gastrulation, we aligned embryos vertically or end on, such that their dorso-ventral axis was visible. We observed that post cellularization, mitochondrial apical transport is enhanced only in the ventral furrow cells. Mitochondrial transport was not seen in the lateral region cells post cellularization. The data indicate that specific relocalization of mitochondria is likely to be by the virtue of Toll-Dorsal signalling in the ventral furrow region and there may be a distinctive functional relevance of apical mitochondria in the ventral furrow cells. We tested whether this pathway influenced mitochondrial transport further.

2.4 Mitochondrial shape is essential for their apical transport

It is known that maintenance of mitochondrial architecture is obligatory for embryonic survival (Chen et al., 2003; Ishihara et al., 2009; Moore et al., 2010). We maternally depleted the levels of fission protein Drp1 and fusion proteins Marf and Opa1 using RNAi or genomic mutations. We also employed a strategy of over-expressing mutant form of proteins.

Optimum knockdown of these proteins was lethal. We found that mitochondria were clustered in drp1 knockdown or mutations. The morphology did not change in Marf or Opa1 knockdown. We followed drp1 mutant: drp1^SG and drp1 RNAi (drp1ⁱ) based on the extent of phenotype and embryonic lethality. We were able to revert the mitochondrial shape from clustered to fragmented when we knocked down opa1 in the background of drp1^SG (drp1^SG; opa1ⁱ). This indicated that both fission and fusion machinery are active in the embryos.

Mitochondria formed large clusters of mitochondria located at the basal regions in drp1^SG and drp1ⁱ. These large clusters of mitochondria failed to migrate apically during

cellularization and gastrulation. We observed a rescue of this transport defect in drp1^SG; opa1ⁱ embryos. Therefore, mitochondrial morphology is essential for their apical transport.

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xvi 2.5 Cellular morphology is altered in drp1^SG embryos.

We then analysed how drp1 mutation may affect the process of cellularization. We visualized membrane extension dynamics during cellularization using tagged Myosin regulatory light chain (Sqh-mCherry). The cellularization ended abruptly and significantly shorter cells were formed in the drp1^SG embryos. The basal contractile rings of these cells did not completely constrict to enclose the cellular compartment. Myosin II localization and activity is required for membrane extension and constriction during cellularization (He et al., 2016; Wenzl et al., 2010; Young et al., 1991). drp1^SG embryos had lowered levels of Sqh, likely leading to membrane extension and constriction defects. Myosin activity is regulated by ROS levels during Drosophila embryo dorsal closure (Muliyil and Narasimha, 2014). In accordance with this study, we found that the level of ROS was reduced in drp1^SG embryos and is likely the cause of Sqh depletion.

2.6 drp1^SG embryos contain shallow ventral furrow with misaligned cells.

Pulsatile Myosin II helps in the apical constriction and invagination of ventral furrow cells (Martin et al., 2009). We found that ventral furrow in drp1^SG embryos was jagged and had misaligned cells. The furrow morphology appeared shallow in the end-on orientation. The furrow did not completely close. Similar to cellularization, Sqh levels were reduced at the apical regions of the ventral furrow cells. This suggests that mitochondrial localization and probably ROS based activity is required for ventral furrow morphogenesis.

2.7 Apical transport of mitochondria during gastrulation depends on Toll-Dorsal signalling Ventral fate is regulated by Toll-Dorsal signalling through a number of transcription factors regulating Myosin II localization at the apical regions of ventral furrow cells (Dawes-Hoang, 2005; Martin et al., 2009; Morisato, 2001). We had observed that mitochondria localize specifically at the apical regions of ventral cells. Also, the lack of mitochondrial transport and activity in drp1^SG embryos led to decreased Myosin activity and distorted ventral furrow morphology. Over-expression of Dorsal in the Drosophila embryos extended the zone of mitochondrial apical transport to almost the entire embryo compared to a restricted ventral region in the WT. Knockdown of a downstream factor fog reduced mitochondrial transport

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xvii at the ventral furrow. Thus based on these experiments, mitochondria operate under the influence of Toll-Dorsal pathway and possibly aid in regulating Myosin II activity.

In summary, mitochondrial distribution is regulated at distinctive stages of embryonic development in Drosophila. Their shape and activity are essential for membrane ingression during syncytial stage and cellularization and ventral furrow ingression during gastrulation.

The study partly demonstrates and proposes the regulation of mitochondria by axis determination Toll-Dorsal pathway in the Drosophila embryogenesis.

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1

Chapter 1 Introduction

1.1 Mitochondria are essential organelles of eukaryotic cells

It is thought that mitochondria have evolved from a symbiont α-proteobacteria species found inside eukaryotic host cells. Mitochondria are semi-autonomous organelles holding significance in aerobic metabolism and production of ATP in cells. They possess their own DNA, obtained from their bacterial form. Apart from the known function of ATP production, their shape and distribution suggest their role in regulating the essential signalling processes of cell. Transportation of mitochondria occurs via microtubules and their localization is necessary for survival and effective functioning of cell. Different kinds of cells demonstrate various shapes of mitochondria. Stem cells and embryos have comparatively smaller and lesser number while on the other hand, differentiated cells can show long shaped ones.

Embryonic development depends on mitochondrial function. Also the literature indicates that any perturbations in the shape of mitochondria affects embryonic patterning and can lead to lethality. The regulation of mitochondrial morphology and its functions in the embryo still remains an information to be uncovered.

1.1.1 Mitochondria produce cellular ATP

As the primary function of mitochondria is ATP production, they are called “the powerhouse of the cell”. The mitochondria are semi-autonomous due to presence of its own DNA and ribosomes, that enable it to produce tRNAs and components of electron transport chain.

The ATP generation occurs by redox cycles of electron transport chain (ETC) proteins which are found in in the inner mitochondrial membrane. This membrane folds to form numerous loops called cristae (Fig. 1.1) (Stroud and Ryan, 2013). Mitochondria play a role in

oxidization of carbon compounds by a chain of redox reactions in ETC, called as the Kreb’s cycle or tricarboxylic acid (TCA) cycle. The ETC involves four protein complexes that transport electrons, acting as electron acceptors and donors. These complexes are NADH dehydrogenase (Complex I), succinate dehydrogenase (Complex II), cytochrome bc1

complex (Complex III) and Cytochrome oxidase (Complex IV). ETC complexes are coupled to ATP synthase complex utilizes the proton gradient across inner mitochondrial membrane to

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2 hydrolyse ADP to ATP in the mitochondrial matrix (Fig. 1.2). Water molecules are produced as a result of reduction of oxygen during electron transfer. 34 ATP molecules are produced during the Kreb’s cycle and oxidative phosphorylation (Ernster and Schatz, 1981).

Respiratory chain complexes also form super-complexes that play a role in raising the efficiency of respiration. This was seen in yeast and mammalian cells (Lapuente-brun et al., 2013; SchaÈgger and Pfeiffer, 2000). Supercomplex assembly is controlled by inner

membrane architecture, in turn by mitochondrial inner membrane fusion protein Opa1 (Cogliati et al., 2013). There is a close association of ATP production and cristae architecture.

The intricate cristae structure brings about assembly of more super complexes and thus increasing ATP generation (Cogliati et al., 2013). The last component of ETC, i.e. ATPase is also central l for maintaining the structure (Saddar et al., 2008).

A

B

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3 Mitochondrial reactive oxygen species (ROS) is produced as a by-product of electron leakage from the ETC, primarily from Complex I and III of the ETC (Fig. 1.2) (Bell et al., 2007; Liu et al., 2002). A number of defence systems are utilized in cells to reduce ROS. Superoxides generated are modified by superoxide dismutase (SOD) to diffusible hydrogen peroxide (H2O2). Glutathione peroxidase silences ROS by oxidizing glutathione. ROS is scavenged in peroxisomes by the catalase enzymes (Ray et al., 2012). Mitochondrial shape and signalling interacts and modifies ROS. This is discussed later on in this section.

Intermembrane Space

Matrix ^ROS ^ROS

ROS

Figure 1.1: Mitochondrial structure. The schematic depicts mitochondrial double membraned architecture. The superfolded inner membrane (yellow) houses ETC. The mitochondrial matrix (blue) consists of DNA, ribosomes and granules (A). An electron micrograph shows mitochondria in Drosphila brain. Electron dense cristae (black arrows) are organized in parallel array (B). Adapted from (Macchi et al., 2013) (B)

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4 1.1.2 Mitochondria undergo fission and fusion

1.1.2.1 Mitochondrial morphology is regulated by fission-fusion machinery

Distinct mitochondrial morphologies have been observed in different cell types and

according to cell requirements. First observation of mitochondrial morphology changes was done about a hundred years ago (Lewis and Lewis, 1915). Authors used light microscopy and hand drawings to describe their observations. Since then, in the last couple of decades mitochondrial fission and fusion have been very well characterized. Mitochondrial

morphology regulators were first found in yeast. A temperature sensitive yeast mutation in the dynamin mgm1 showed defects in the mitochondrial DNA maintenance (Jones and Fangman, 1992). Similar phenotype was observed with mutation in mitochondrial outer membrane GTPase protein Fzo in Drosophila sperms (Hales and Fuller, 1997). Mutations in Fzo lead to mitochondrial fragmentation (Hermann et al., 1998) and loss of mtDNA copies from the mitochondrial fragments (Rapaport et al., 1998). Mitochondria were visualized with mitochondrial GFP tag and mitochondrial dye: Mitotracker using an epifluorescence microscope (Rapaport et al., 1998). Mgm1 was later characterized as the mitochondrial inner membrane fusion protein and was shown to co-localize with mitochondria (Wong et al., 2000). The protein homolog was also found to be involved in neuropathies and was named as Optic atrophy 1 or Opa 1 in mammals and Drosophila (Delettre et al., 2000) (Fig.

1.3 A). The mammalian orthologs of Fzo, Mitofusins (Mfn1 and Mfn2) (Fig. 1.3 A) (Santel and Fuller, 2000) were shown to have partially redundant functions and both were shown to be essential for mouse embryonic development (Chen et al., 2003). Mitochondria assembly

Figure 2.1 ATP is genetated by mitochondrial electron transport chain. The ETC

complexes reside in the mitochondrial inner membrane (grey). The electrons (green) are relayed from complex I to complex IV by oxidizing NADH at Complex I (light blue), FADH2 at Complex II (light green), reduction and oxidation of Cytochrome C (yellow) by Complex III (light yellow) and IV (light orange) respectively. Protons (red) are pumped in the intermembrane space by Complex I, III and IV. Complex V (ATP synthase, pink) uses proton flux to generate ATP (blue) in the matrix. ROS is generated by leaky electrons from Complex I and III. Adapted from (Keane et al., 2011).

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5 regulating factor (Marf), another outer membrane fusion protein in Drosophila, along with Opa1 has been shown to be essential for cardiomyocyte function (Dorn et al., 2011).

Mitochondrial fission protein, Dnm1p, was discovered in a yeast genetic screen and was found to be associated with fission sites (Mozdy et al., 2000; Otsuga et al., 1998; Shaw and Nunnari, 2002). The protein, most commonly known as Dynamin related protein 1 (Drp1) is conversed in most eukaryotes and resulted in mitochondrial fragmentation (Labrousse et al., 1999; Smirnova et al., 1998a). Using in vitro EM studies, it was observed in the yeast that Dnm1 can oligomerize around tubules of the size of mitochondria in vitro (Ingerman et al., 2005), similar to the activity of dynamin at the endocytic vesicles. Drp1 is present largely in cytoplasm and localizes to the mitochondria upon fission cues. Outer membrane scaffolding protein Fis1 (or Fis1p in yeast) is required for binding of Drp1on the mitochondria (Mozdy et al., 2000) which then results in membrane scission. More recently additional adapter

proteins were found to play a role in recruit Drp1 on the mitochondrial outer membrane.

Mdv1 (Koirala et al., 2010; Zhang and Chan, 2007) and Caf4p (Griffin et al., 2005) are proteins that bind to Fis1 and facilitate Dnm1 binding in yeast. In higher eukaryotes, since there are not mdv1 homologs found, other adapters seem to have taken over this function.

Mitochondrial fission factor (Mff) binds to Drp1 independent of Fis1 and is responsible for mitochondrial fission in mammalian cells (Otera et al., 2010) (Fig. 1.3 A). Proteins like Mid49, GDAP1 and Mid51/Mief (Loson et al., 2013) have been shown to recruit Drp1 and cause mitochondrial fragmentation as well, indicating that there may be redundancy in the

regulation of mitochondrial fission in higher eukaryotes such as mammals. The final scission of the membrane after oligomerization of Drp1 is done by the bar domain protein

Endophilin B1 (Karbowski et al., 2004).

The activity of fission and fusion proteins is regulated by a number of post translational modifications. Fission protein Drp1 is known to be modified with phosphorylation, SUMOylation, Ubiquitination and S-nitrosylation. Mitochondrial fission during mitosis in mammalian cells is promoted by phosphorylation of Drp1 at GTPase effector domain (GED) by cyclic-AMP dependent kinase A (PKA) in calcium dependent manner (Chang and

Blackstone, 2007; Cribbs and Strack, 2007; Taguchi et al., 2007). This is mediated by RalA, effector RalB and Aurora kinase A in an Mff dependent manner (Kashatus et al., 2011). Ser 637 is a putative phosphorylation site in Drp1 conserved in metazoans and is regulated by

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6 multiple mechanisms. Phosphorylation and dephosphorylation at this site by PKA and

calcineurin respectively regulate localization of Drp1 on the mitochondria (Cereghetti et al., 2008; Taguchi et al., 2007). Drp1 is shown to be SUMOylated by mitochondrially anchored protein ligase (MAPL) and leads to mitochondrial fragmentation (Braschi et al., 2009). The SUMOylation is dependent on BAK/BAX protein kinases and could be essential to initiate apoptosis in cells (Wasiak et al., 2007). SUMO protease activity of SENP5 inhibits Drp1 activity and makes mitochondrial network more fused (Zunino et al., 2007). Regulation of Drp1 activity is also achieved by ubiquitination. Loss of function variant of another

mitochondrial E3 ubiquitin ligase, MARCH5 resulted in mitochondrial elongation (Karbowski et al., 2007). MARCH5 was later shown to play a role in clearing misfolded SOD protein from mitochondria and protecting the cells from oxidative damage (Yonashiro et al., 2009).

Mitochondrial fission Mitochondrial fusion

A

Fission

Fusion B

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7 Observations through EMs have suggested proximal localization of mitochondrial outer membrane (OMM) and ER (Csordás et al., 2006). Mitochondria associated ER membranes (MAMs) have been shown to be essential for lipid synthesis (Stone and Vance, 2000; Vance, 1990), ATP synthesis, trafficking and calcium regulation and this depends on the distance between the ER and the OMM (Csordás et al., 2006, 2010; Rowland and Voeltz, 2012).

Localization of mitochondrial morphology proteins at MAMs regulate mitochondrial shape, and thereby the function in the vicinity of ER. Studies using E.M. and super-resolution microscopy (STORM) demonstrated the presence of ER tubule loops winding around mitochondrial fission sites allowing constriction of the mitochondria prior to Drp1

recruitment (Friedman et al., 2011; Shim et al., 2012). Inverted formin 2 (INF2), a regulator of actin polymerization and depolymerization (Chhabra and Higgs, 2006) localizes at the ER- mitochondria contact sites, where it polymerizes F-actin around mitochondria to cause a constriction initiation such that binding of Drp1 at these sites is enhanced (Korobova et al., 2013). Microtubule motors dynein and dynactin complexes transport Drp1 to the

mitochondria (Varadi et al., 2004). A recent study suggests a relationship between microtubule polymerization and mitochondrial size in yeast (Mehta et al., 2017).

Therefore, mitochondria can acquire a variety of shapes and sizes in cells by activity and regulation of fission fusion machinery. Remodelling of mitochondria is essential for

Figure 1.3 Mitochondria undergo fusion and fission. Mitochondrial fusion is regulated by Mfn1 and Mfn2 located on the mitochondrial outer membrane (A, fusion, blue).

Transmembrane protein Opa1 is required from inner mitochondrial membrane fusion (A, fusion, orange). Cytosolic Drp1 (A, fission, yellow) binds to mitochondrial

membrane localized Fis1 (A, fission, orange) upon activation. Active Drp1 oligomerizes around the mitochondrion to drive fission (A, fission). Fusion is associated with

increased ATP output, oxygen consumption and membrane potential and vice versa with fission (A). Schematic is adapted from (Chiong et al., 2014) (A) Fluorescence imaging of mitochondria in yeast cells shows conversion of mitochondrial network to a hyperfused state upon fusion and punctate structures upon fragmentation (B). Adapted from (Mozdy et al., 2000) (B).

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8 physiological processes in cells. A constant cycling between fusion and fission is said to be present in mammalian cells (Fig. 1.4 A, B). Mitochondrial biogenesis occurs by creating new mitochondria from an existing mitochondrial pool by fission (Goto et al., 2006; Marti et al., 2009) (Fig. 1.4 C). Association of mitochondria is enhanced on fusion and maintains calcium homeostasis in the cells (Grimm, 2012) (Fig. 1.4 A). Apoptotic cues lead to mitochondrial fragmentation and release of Cytochrome C which activates the caspases (Estaquier and Arnoult, 2007; Kluck and Newmeyer, 2013) (Fig 1.4 E). Mitochondrial fission and

depolarization of the membrane potential lead to mitophagy (Burman et al., 2017) (Fig. 1.4 D). Additionally, there is a link between mitochondrial architecture and their metabolic capacity and ROS generation (Chan, 2006). Together, mitochondrial architecture coupled with the metabolic function has been studied in the context of cellular signalling pathways as discussed further.

Calcium signalling

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9 1.1.2.2 Mitochondrial morphology and metabolism are interrelated.

There is a close connection between mitochondrial shape and their function. Mitochondrial shape is regulated in cells based on energy requirements, cell type and resources (Kuznetsov et al., 2009). Early embryos (Motta et al., 2000; Sathananthan and Trounson, 2000) and stem cells (Chen et al., 2012) contain small, dispersed mitochondria. Differentiated cells, such as muscles (Skulachev, 2001), rat cardiomyocytes (Ong and Hausenloy, 2010) ,

pancreatic cells (Kuznetsov et al., 2010), Drosophila oocyte main body follicle cells (Mitra et al., 2012) contain an intercalated mitochondrial network. These interconnected

mitochondrial networks are said to be electrically coupled, have efficient regulation

membrane potential to provide for high energy requirements. Mitochondrial structures are regulated based on cellular states. Growth phase yeast cells which depend on aerobic respiration have elaborate mitochondria (Egner et al., 2002; Hoffmann and Avers, 1973). A shift to glycolytic, fermentation state resolves the mitochondrial network (Jakobs et al., 2003). Comparably, mitochondria in G1-S phase of mammalian cells are more reticular compared to M phase, where they fragment to ensure proper distribution between daughter cells (Mitra et al., 2009). Mitochondria hyperfuse and produce more ATP upon presentation of stress stimuli to the cells (Tondera et al., 2009). Mitochondrial hyperfusion may be necessary to protect cells from degeneration (Chen et al., 2007). Mitochondrial fusion by inhibition of fission protein Drp1 during starvation stress via mTOR signalling is also proposed to have similar protective role (Gomez et al., 2011; Rambold et al., 2011).

Mitochondrial inner membrane houses the ETC complexes and maintenance of cristae structure with the help of inner membrane fusion protein Opa1 essential for ETC

Figure 1.4: Physiological roles of mitochondrial morphology. Mitochondrial fusion is regulated by Opa1, Mfn1 and Mfn2 (A). Calcium based signalling occurs at the ER – mitochondria interphase (A). Mitochondrial fission is promoted by the Drp1 (B).

Biogenesis of mitochondria requires fusion and promotes cell growth and metabolism (C).Mitochondrial fission depolarizes mitochondria leading to mitophagy by lysosome fusion followed by degradation (D). Apoptosis is promoted by Bcl-2 family proteins.

Apoptosis requires mitochondrial fission for Cytochrome C release (E). Schematic adapted from (Boland et al., 2013).

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10 supercomplex assembly and function (Cogliati et al., 2013; SchaÈgger and Pfeiffer, 2000). A number of studies associate increased mitochondrial fusion with OXPHOS activity elevation (Chan, 2006), membrane potential (Chen et al., 2003; Tomer et al., 2018) and reduction in ROS. On the other hand, smaller mitochondria are associated with minimal ATP production (Chen and Chan, 2005; Westermann, 2012). Mitochondrial fusion ensures maintenance of mtDNA. Depletion of mitochondrial fusion proteins in yeast, neurons and mouse skeletal muscles led to mutations and loss of mtDNA (Chen et al., 2010, 2007; Jones and Fangman, 1992; Merz and Westermann, 2009). mtDNA is required for synthesis of ETC components . The loss of mtDNA in the fusion mutants leads to further reduction in the mitochondrial metabolic output. Increased fission shortens yeast life span (Scheckhuber et al., 2011).

Conversely, mitochondrial fission is aids removal of damaged mitochondria and maintains healthy, productive mitochondria in the system (Twig et al., 2008). Loss of fission increased life span of the yeast (Scheckhuber et al., 2006). Increased fusion also extended the lifespan of C. elegans (Chaudhari and Kipreos, 2017).

Mitochondrial morphology is regulated based on cellular resources, ATP levels and ETC activity. Inhibition of ETC by dissipation of the membrane potential leads to mitochondrial fragmentation in yeast and mammalian cells (Ishihara et al., 2003; Legros and Lombès, 2002; Meeusen et al., 2004). Uncouplers cause Opa1 cleavage and calcium mediated activation of Drp1 to cause mitochondrial fission (Cereghetti et al., 2008; Cribbs and Strack, 2007). Decrease in the ATP concentration activate AMP-Kinase (AMPK) pathway that leads to increased mitochondrial fragmentation followed by cell death (Toyama et al., 2016).

1.1.3 Microtubules regulate mitochondrial transport

Mitochondrial transport has been extensively analysed in neuronal cells and is essential acquiring mitochondria from the cell body (Saxton and Hollenbeck, 2012). Mitochondria are transported bi-directionally on microtubules on Dynein and Kinesin motors (Saxton and Hollenbeck, 2005) (Fig. 1.5). Kinesin-1 motors primarily carry mitochondria in the

anterograde direction, or to the microtubule plus ends, whereas Dynein carries them in the opposite direction. The transport is facilitated by binding and activation of microtubule motor with adapters such as Dynactin and Milton (Koutsopoulos et al., 2010; Martin et al., 1999; Steffen et al., 1997). Mitochondrial Rho-like GTPases (Miro1 and Miro2) facilitate

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11 mitochondrial binding to microtubule motor adapters and are responsible for carrying mitochondria to either plus or minus ends of microtubule motors (López-Doménech et al., 2018; Saotome et al., 2008). Neurotransmitter mediated Ca⁺² signalling can initiate

mitochondrial transport in axons (Mironov, 2006; Rintoul and Reynolds, 2010; Saotome et al., 2008). Studies from Drosophila demonstrate that Miro (Fransson et al., 2006a) and Milton (Glater et al., 2006; Górska-Andrzejak et al., 2003; Stowers et al., 2002) act as Ca⁺² sensors and form a complex with Kinesin-1 which mediates mitochondrial transport (Glater et al., 2006; Wang and Schwarz, 2009). Milton based mitochondrial transport is essential for mitochondrial inheritance in Drosophila (Cox and Spradling, 2006).

Reduction in ATP levels or hypoxia at the axons stimulates mitochondrial transport by AMPK and HIF-1α signalling respectively (Li et al., 2009; Tao et al., 2014). A selective population of mitochondria with higher membrane potential are transported in the anterograde direction (Amiri and Hollenbeck, 2008; Miller and Sheetz, 2004). Mitochondria with lower membrane potential are carried to the neuronal cell bodies (Miller and Sheetz, 2004). Depolymerization of mitochondria using ETC inhibitors halts their transport in either direction (Baqri et al., 2009).

Figure 1.5. Mitochondrial transport of microtubules. Microtubule (royal blue) motors Kinesin-1 (yellow) and Dynein (green) carry mitochondria in anterograde and retrograde direction respectively. They bind to mitochondria by interacting with mitochondrion specific adapters: Milton (orange) and Miro (blue). Reproduced from (Schwarz, 2013).

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12 Defects in mitochondrial fission and fusion render them immobile in neurons (Chen et al., 2003; Li et al., 2004; Verstreken et al., 2005). Additionally, Miro and Milton over-expression can induce mitochondria fusion affecting mitochondrial transport (Fransson et al., 2006b;

Koutsopoulos et al., 2010; Saotome et al., 2008). Lack of mitochondrial transport results in severe consequent neurodegenerative disorders including Parkinson’s, Alzheimer’s, ALS, Charcot-Marrie-Tooth type 2A and Schizophrenia (Lovas and Wang, 2013). Thus, it is essential to examine the mitochondrial transport dynamics in a developmental system to track down the mechanism leading to disorders.

1.1.4 Mitochondria are involved in signalling

Mitochondrial regulation of signalling pathways is an emerging field of research. The signalling pathways are more or less conserved across species. Role of mitochondrial shape and metabolism in the regulation of cell death, cell growth and differentiation and cell cycle has been explored in the field.

1.1.4.1 Mitochondria are key components of apoptosis

The earliest discovered signalling cascade to interact with mitochondria was apoptosis machinery (Liu et al., 1996). Apoptosis cues lead to Drp1 dependent mitochondrial

fragmentation (Karbowski et al., 2002). Mitochondrial fragmentation is essential for release of cytochrome C to the cytoplasm and activation of the downstream caspase cascade (Estaquier and Arnoult, 2007; Frank et al., 2001). Also, inhibition of Drp1 leads to delayed apoptotic signalling (Frank et al., 2001). Release of Opa1 and breakdown of cristae structure by mitochondrial membrane permeability transition is also required from Cytochrome C release (Zhang et al., 2008a) and downregulation of Opa1, leading to mitochondrial

fragmentation leads to spontaneous apoptosis. Bax protein, a regulator of caspase cascade (Youle and Strasser, 2008), localizes onto mitochondria on apoptotic stimuli (Dewson and Kluck, 2009) and triggers cytochrome C release (Kluck and Newmeyer, 2013; Walensky and Gavathiotis, 2011). The apoptotic signals localize more on the ER associated mitochondria (Csordás et al., 2006). This association increases upon Ca⁺² exchange between the two organelles (Weaver et al., 2004). Pro-apoptotic proteins like Bax and Bak increase the Ca⁺² dependent mitochondria (Oakes et al., 2005) and ER interaction where as anti-apoptotic Bcl- 2 and Bcl-XL release Ca⁺²(Chen and Dickman, 2004; Pinton et al., 2000; White et al., 2005).

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13 1.1.4.2 Mitochondria regulate cellular growth and differentiation

Activation of epidermal growth factor (EGFR) triggers cellular growth and is correlated with cancer progression (Sharma et al., 2007). EGFR localizes to the mitochondrial outer

membrane (Boerner et al., 2004; Che et al., 2015; Demory et al., 2009; Zhang et al., 2005) and regulates mitochondrial dynamics (Che et al., 2015; Mitra et al., 2012). EGFR causes Drp1 dependent mitochondrial fragmentation in Drosophila follicle cells and activates Notch pathway (Mitra et al., 2012) through regulation of mitochondrial membrane potential (Tomer et al., 2018). Likewise, dominant negative mutation of ERK, a downstream kinase in EGFR pathway, led to depletion of membrane potential and ATP levels in human alveolar macrophages (Monick et al., 2008) and ATP synthase depletion in astrocytes (Yung et al., 2004). Notch pathway increases mitochondrial fusion by prevention of mitochondrial fragmentation by Akt pathway (Perumalsamy et al., 2010). Mitochondrial fusion increases with Yorkie activation, leading to cell proliferation in Drosophila pupal eye discs and mammalian cell (Nagaraj et al., 2012; Ohsawa et al., 2012). On the other hand, mitochondrial fission increases cell proliferation in hepatocellular carcinomas in p53 dependent manner (Zhan et al., 2016).

Mitochondrial morphology is also regulated temporally during cell cycle. Mitochondrial hyperfusion during G1-S phase of cell cycle regulates energy output and is required for maintenance of cyclin E during the S phase (Mitra et al., 2009; Parker et al., 2015). Drp1 mediated mitochondrial fragmentation is essential for mitochondrial segregation in daughter cells (Mitra et al., 2009).

In summary, mitochondria interact with major cellular growth and differentiation pathways and the mechanisms of these interactions are yet to be uncovered.

1.1.4.3 Mitochondrial activity and ROS based signalling

Maintaining energy homeostasis is essential for cellular survival. AMP-activated protein kinase (AMPK) is activated by liver kinase B1 (LKB1) when the AMP:ATP or ADP:ATP ratios increase in the cells and inhibits cellular activities high energy requirements (Hardie et al., 2006). Other modes of mitochondrial dysfunction include signalling by mitochondrial ETC intermediates (Bohovych and Khalimonchuk, 2016). Depletion of mitochondrial activity by

Role of mitochondrial dynamics and function in Drosophila embryo morphogenesis